Mapping functional domains on the acetylcholine receptor using monoclonal antibodies and small angle X-ray diffraction

54 MONOCLONAL ANTIBODIES AGAINST TWO PEPTIDE SEQUENCES OF THE ACETYLCHOLINE RECEPTOR DISTINGUISH BETWEEN TOXIN BINDING AND CHOLINERGIC LIGAND BINDING

Robert H. Fairclough, Mirta Mihovilovic, Ferenc J. Kezdy and David P. Richman Departments of Neurology and Biochemistry and Molecular Bilogy, The University of Chicago and Division of Neurology Duke University

To analyze the cholinergic bindingsite of the nicotinic acetylcholine receptor (AChR), with the long-term goal of identifying the primary-structure and tertiarystructure locations of this site, we have produced monoclonal antibodies (n~Abs) against two synthetic peptides from a candidate region of the alpha subunit. With the f i r s t peptide, al27-al45, as immunogen, 9 rat n~Abs were obtained that bound to free (unconjugated) peptide and 4 that bound to native AChR. Three of the anti-peptide n~Abs also bound native AChR. For the second peptide, which is more hydrophobic, a187=a205, the immunogen was peptide conjugated to keyhold limpet hemocyanin. The antigen employed in the antibody assay was peptide conjugated to bovine serum albumin. Two mAbs were obtained from this latter immunization, one that bound to conjugated peptide and to native AChR and one that bound only to native AChR. The binding to membrane-bound AChR of a mAb recognizing each peptide was studied in greater detail. For ~Ab KM3-7EG, that binds to the f i r s t peptide, mAb binding stoichiometry to the AChRwas diminished by alpha bungarotoxin (aBgTx) and carbamylcholine (carb) without an apparent change in a f f i n i t y , whereas n~Ab binding stoichiometry and a f f i n i t y were both decreased by d-tubocurarine {tubo). For the AChR-crossreactive n~Abagainst the second peptide, mAb KNI-5G, aBgTx had no effect on the binding to AChR, while carb and tubo decreased mAb stoichiometry as well as the a f f i n i t y . In summary the two epitopes on the AChR recognized by the mJ~bs directed against each of the two peptides were both significantly altered by tubo, but were d i f f e r e n t i a l l y affecteo by aBgTx and carb.

Mapping Functional Domains on the Acetylcholine Receptor Using Monoclonal Antibodies and Small Angle X-ray Diffraction Robert H. Fairclough and David P. Richman Department of Neurology, University of Chicago, Chicago, I l l i n o i s 60637 A library of anti-acetylcholine receptor (anti-AChR) monoclonal antibodies (n~Ab) has been produced whose members bind to epitopes that are d i f f e r e n t i a l l y accessible in several AChR states. We have conducted small angle X-ray diffraction experiments on membranes that have been treated with mAbs and oriented ultracentrifugally. This allows localization of the mAb relative to characteristic features of the membrane bilayer. Electron density profiles normal to the membrane plane have been extracted from meridional diffraction amplitudes for membrane complexes with several ~LAbs that disrupt one or more receptor functions, including carbamylcholine (carb) binding, d-tubocurarine (tubo) binding, a-bungarotoxin (a-BgTx) binding, and agonist induced ion fluxing. The electron density profile obtained for membranes treated with mAb 383C contains extra

5~ electron density centered midway between the top of the AChR synaptic protrusion and the extracellular phosphate head groups of the bilayer. This antibody binds 2:1 to the AChR, inhibiting the binding of a-BgTx and carb. Furthermore, binding of a-BgTx or carb to the AChR blocks the binding of mAb 383C to the receptor. We suspect that the added electron density covers a cholinergic binding site of the AChR.

SEMLIKI

FOREST

Thomas'

Hospital,

VIRUS London,

INDUCED,

IMMUNE H.E.

MEDIATED DEMYELINATION. J.K. Webb. Neurovirology U r i t , St

UK.

Infection of adult m i c e w i t h the a v i r u l e n t A 7 ( 7 4 ) s t r a i n of the encephalitic alphavirus, S e m l i k i F o r e s t v i r u s r e s u l t s in a t r a n s i e n t viFaemia, infection c,f t h e CNS, a p l e o c y t o s i s , blood-brain barrier changes, abnormal VER's, and a demyelinatir,g encephalomyelitis with involvement of the optic nerves. EM studies indicate preservation of the axons in the demyelinated lesions ar,d demonstrate the close association of lymphoblastic cells, astrocytes and m y e l i n . Athymic "InLide ~ m i c e develop a life-long i n f e c t i o n of t h e CNS, a n d s t u d i e s indicate that t h e v i r u s a l s o p e r s i s t s in n o r m a l i m m u n o c o m p e t e n t adult mice. Viral persistence cannot be demonstrated by infectivity assays, except following immunosuppression by c y c l o p h o s p h a m i d e , but is readily demonstrated by immunocytochemistry. Immunosuppression experiments, and experiments in athymic ~ n u d e ~, N K - c e l l d e f i c i e n t "beige ~ , and mice depleted of complement, indicate that the d e m y e l inat ion is immune mediated and directly dependant upon sensitised T-lymphocytes. It is n o t c l e a r w h e t h e r the i m m u n e m e d i a t e d demyelination is a response to v i r a l or a u t o a n t i g e n s on the s u r f a c e of the myelin or its supporting cells the oligodendrocytes. Mice infected with the virus develop anti-glycolipid antibodies, w h i c h may be importa~nt in the pathogenesis of the demyelination. Monoclonal antibodies raised to the v i r u s r e a c t w i t h a g l y c o l i p i d c o m p o n e n t of CNS myelin.

SYNERGISM IN THE PATHOGENESIS OF EAE INDUCED BY AN MBP-SPECIFIC T CELL L I N E A N D M O N O C i X ) N A L ANTIB()DIES T O G A L A C T O - C E R E I 3 R O S I D E O R T O ~% M Y E L I N OLIGODENDR(~LiAL G L Y C O i # R O T E I N . ' " W. FIERZ i, K. H E I N I N G E R 2, B. S C H A E F E R 2, K.V. T O Y K A 2, CH. L I N I N G T O N ~ a n d H. L A S S M A N N 4 1 2 a 4

S e c t i o n o f Clinical I m m u n o l o g y , U n i v e r s i t y H o s p i t a l , Ziirich, S w i t z e r l a n d D e p a r t m e n t of N e u r o l o g y , U n i v e r s i t y of D ( i s s e l d o r f , FRG D e p a r t m e n t of Medicine, U n i v e r s i t y of Wales, Cardiff, Wales, GB D e p a r t m e n t of Neurology, U n i v e r s i t y of Vienna, A u s t r i a

In the L E W I S rat, T cells specific for myelin basic protein (MBP) induce an acute E A E which is mainly inflammatory without gross demyelination. O n the other hand, it has recently been demonstrated that a monoclonal antibody (8-18-C5) specific for a 54kD myelin oligodendroglial glycoprotein (MOG) mediates demyelination in vivo if given access to the C N S b y intrathecal administration or through an inflammatory opening of the blood brain barrier during the course of a passively transferred E A E (Linington et al., submitted}. Here, w e describe similar findings with a monoclonal antibody to galacto-cerebroside (GC} leading to